Many peptides and proteins self-assemble into amyloidfibrils. Examples include mammalian and fungal prion proteins, polypeptides associated with human amyloid diseases, and proteins that may have ...biologically functional amyloid states. To understand the propensity for polypeptides to form amyloid fibrils and to facilitate rational design of amyloid inhibitors and imaging agents, it is necessary to elucidate the molecular structures of these fibrils. Although fibril structures were largely mysterious 15 years ago, a considerable body of reliable structural information about amyloid fibril structures now exists, with essential contributions from solid state nuclear magnetic resonance (NMR) measurements. This Account reviews results from our laboratories and discusses several structural issues that have been controversial. In many cases, the amino acid sequences of amyloid fibrilsdo not uniquely determine their molecular structures. Self-propagating, molecular-level polymorphism complicates the structure determination problem and can lead to apparent disagreements between results from different laboratories, particularly when different laboratories study different polymorphs. For 40-residue β-amyloid (Aβ1–40) fibrils associated with Alzheimer's disease, we have developed detailed structural models from solid state NMR and electron microscopy data for two polymorphs. These polymorphs have similar peptide conformations, identical in-register parallel β-sheet organizations, but different overall symmetry. Other polymorphs have also been partially characterized by solid state NMR and appear to have similar structures. In contrast, cryo-electron microscopy studies that use significantly different fibril growth conditions have identified structures that appear (at low resolution) to be different from those examined by solid state NMR. Based on solid state NMR and electron paramagnetic resonance (EPR) measurements, the in-register parallel β-sheet organization found in β-amyloid fibrils also occurs in many other fibril-forming systems. We attribute this common structural motif to the stabilization of amyloid structures by intermolecular interactions among like amino acids, including hydrophobic interactions and polar zippers. Surprisingly, we have recently identified and characterized antiparallel β-sheets in certain fibrils that are formed by the D23N mutant of Aβ1–40, a mutant that is associated with early-onset, familial neurodegenerative disease. Antiparallel D23N-Aβ1–40 fibrils are metastable with respect to parallel structures and, therefore, represent an off-pathway intermediate in the amyloid fibril formation process. Other methods have recently produced additional evidence for antiparallel β-sheets in other amyloid-formation intermediates. As an alternative to simple parallel and antiparallel β-sheet structures, researchers have proposed β-helical structural models for some fibrils, especially those formed by mammalian and fungal prion proteins. Solid state NMR and EPR data show that fibrils formed in vitro by recombinant PrP have in-register parallel β-sheet structures. However, the structure of infectious PrP aggregates is not yet known. The fungal HET-s prion protein has been shown to contain a β-helical structure. However, all yeast prions studied by solid state NMR (Sup35p, Ure2p, and Rnq1p) have in-register parallel β-sheet structures, with their Gln- and Asn-rich N-terminal segments forming the fibril core.
Yeast and Fungal Prions Wickner, Reed B
Cold Spring Harbor perspectives in biology,
2016-Sep-01, 2016-09-00, 20160901, Letnik:
8, Številka:
9
Journal Article
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Yeast and fungal prions are infectious proteins, most being self-propagating amyloids of normally soluble proteins. Their effects range from a very mild detriment to lethal, with specific effects ...dependent on the prion protein and the specific prion variant ("prion strain"). The prion amyloids of Sup35p, Ure2p, and Rnq1p are in-register, parallel, folded β-sheets, an architecture that naturally suggests a mechanism by which a protein can template its conformation, just as DNA or RNA templates its sequence. Prion propagation is critically affected by an array of chaperone systems, most notably the Hsp104/Hsp70/Hsp40 combination, which is responsible for generating new prion seeds from old filaments. The Btn2/Cur1 antiprion system cures most URE3 prions that develop, and the Ssb antiprion system blocks PSI+ generation.
Anti-prion systems in yeast Wickner, Reed B.
Journal of biological chemistry/The Journal of biological chemistry,
02/2019, Letnik:
294, Številka:
5
Journal Article
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Yeast prions have become important models for the study of the basic mechanisms underlying human amyloid diseases. Yeast prions are pathogenic (unlike the Het-s prion of Podospora anserina), and most ...are amyloid-based with the same in-register parallel β-sheet architecture as most of the disease-causing human amyloids studied. Normal yeast cells eliminate the large majority of prion variants arising, and several anti-prion/anti-amyloid systems that eliminate them have been identified. It is likely that mammalian cells also have anti-amyloid systems, which may be useful in the same way humoral, cellular, and innate immune systems are used to treat or prevent bacterial and viral infections.
and URE3 are prions of
based on amyloids of Sup35p and Ure2p, respectively. In normal cells, antiprion systems block prion formation, cure many prions that arise, prevent infection by prions, and ...prevent toxicity of those prions that escape the other systems. The
,
, and
single mutants each develop
at 10- to 15-fold, but the triple mutant spontaneously generates
at up to ∼5,000-fold the wild-type rate. Most such
variants are cured by restoration of any one of the three defective antiprion systems, defining a previously unknown type of
variant and proving that these three antiprion systems act independently. Generation of
variants stable in wild-type cells is also increased in
strains 25- to 500-fold. Btn2 and Cur1 each cure 90% of URE3 prions generated in their absence, but we find that
or
diminishes the frequency of
generation in an otherwise wild-type strain. Most
isolates in a wild-type strain are destabilized on transfer to a
or
host. Single
or
mutants show the effects of
or
but not
or
strains. The disparate action of Btn2 on URE3 and
may be a result of
's generally higher seed number and lower amyloid structural stability compared with URE3. Thus, prion generation is not a rare event, but the escape of a nascent prion from the surveillance by the antiprion systems is indeed rare.
The yeast prion PSI+ is a self-propagating amyloid of the translation termination factor, Sup35p. For known pathogenic prions, such as PSI+, a single protein can form an array of different amyloid ...structures (prion variants) each stably inherited and with differing biological properties. The ribosome-associated chaperones, Ssb1/2p (Hsp70s), and RAC (Zuo1p (Hsp40) and Ssz1p (Hsp70)), enhance de novo protein folding by protecting nascent polypeptide chains from misfolding and maintain translational fidelity by involvement in translation termination. Ssb1/2p and RAC chaperones were previously found to inhibit PSI+ prion generation. We find that most PSI+ variants arising in the absence of each chaperone were cured by restoring normal levels of that protein. PSI+ variants hypersensitive to Ssb1/2p have distinguishable biological properties from those hypersensitive to Zuo1p or Ssz1p. The elevated PSI+ generation frequency in each deletion strain is not due to an altered PIN+, another prion that primes PSI+ generation. PSI+ prion generation/propagation may be inhibited by Ssb1/2/RAC chaperones by ensuring proper folding of nascent Sup35p, thus preventing its joining amyloid fibers. Alternatively, the effect of RAC/Ssb mutations on translation termination and the absence of an effect on the URE3 prion suggest an effect on the mature Sup35p such that it does not readily join amyloid filaments. Ssz1p is degraded in zuo1Δ psi- cells, but not if the cells carry any of several PSI+ variants. Our results imply that prions arise more frequently than had been thought but the cell has evolved exquisite antiprion systems that rapidly eliminate most variants.
The yeast prion PSI+ is a self-propagating amyloid of Sup35p with a folded in-register parallel β-sheet architecture. In a genetic screen for antiprion genes, using the yeast knockout collection, ...UPF1/NAM7 and UPF3, encoding nonsense-mediated mRNA decay (NMD) factors, were frequently detected. Almost all PSI+ variants arising in the absence of Upf proteins were eliminated by restored normal levels of these proteins, and PSI+ arises more frequently in upf mutants. Upf1p, complexedwith Upf2p and Upf3p, is a multifunctional protein with helicase, ATP-binding, and RNA-binding activities promoting efficient translation termination and degradation of mRNAs with premature nonsense codons. We find that the curing ability of Upf proteins is uncorrelated with these previously reported functions but does depend on their interaction with Sup35p and formation of the Upf1p–Upf2p–Upf3p complex (i.e., the Upf complex). Indeed, Sup35p amyloid formation in vitro is inhibited by substoichiometric Upf1p. Inhibition of PSI+ prion generation and propagation by Upf proteins may be due to the monomeric Upf proteins and the Upf complex competing with Sup35p amyloid fibers for available Sup35p monomers. Alternatively, the association of the Upf complex with amyloid filaments may block the addition of new monomers. Our results suggest that maintenance of normal protein–protein interactions prevents prion formation and can even reverse the process.
Suicidal [PSIâº] is a lethal yeast prion McGlinchey, Ryan P; Kryndushkin, Dmitry; Wickner, Reed B
Proceedings of the National Academy of Sciences - PNAS,
03/2011, Letnik:
108, Številka:
13
Journal Article
Recenzirano
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PSI⺠is a prion of the essential translation termination factor Sup35p. Although mammalian prion infections are uniformly fatal, commonly studied PSI⺠variants do not impair growth, leading to ...suggestions that PSI⺠may protect against stress conditions. We report here that over half of PSI⺠variants are sick or lethal. These "killer PSIâºs" are compatible with cell growth only when also expressing minimal Sup35C, lacking the N-terminal prion domain. The severe detriment of killer PSI⺠results in rapid selection of nonkiller PSI⺠variants or loss of the prion. We also report variants of URE3, a prion of the nitrogen regulation protein Ure2p, that grow much slower than ure2Î cells. Our findings give a more realistic picture of the impact of the prion change than does focus on "mild" prion variants.
The PSI⁺ prion of Saccharomyces cerevisiae is a self-propagating amyloid form of Sup35p, a subunit of the translation termination factor. Using solid-state NMR we have examined the structure of ...amyloid fibrils formed in vitro from purified recombinant$Sup35^{1-253}$, consisting of the glutamine- and asparagine-rich Nterminal 123-residue prion domain (N) and the adjacent 130-residue highly charged M domain. Measurements of magnetic dipole-dipole couplings among$^{13}C$nuclei in a series of Sup35NM fibril samples,$^{13}C-labeled$at backbone carbonyl sites of Tyr, Leu, or Phe residues or at side-chain methyl sites of Ala residues, indicate intermolecular$^{13}C-^{13}C$distances of ≈0.5 nm for nearly all sites in the N domain. Certain sites in the M domain also exhibit intermolecular distances of ≈0.5 nm. These results indicate that an in-register parallel β-sheet structure underlies the PSI⁺ prion phenomenon.
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